The venom of black widow spiders (genus Latrodectus) contains a potent neurotoxin, a-latrotoxin, that evokes mass release of transmitters from vertebrate neurosecretory cells. Though capable of causing severe envenomation in humans, a-latrotoxin also plays an instrumental role in the study of vertebrate neurotransmitter release. Biochemical characterization of one Eurasian species'(L. tredecimguttatus) venom shows that in addition to a-latrotoxin, it contains other neurotoxins that are similar to a-latrotoxin in protein sequence, but which selectively affect invertebrates. Together, a-latrotoxin and these invertebrate toxins are considered functionally distinct members of a common gene family collectively known as latrotoxins. While evidence suggests that latrotoxins may occur in other Latrodectus (31 species) and related species, latrotoxin diversity is virtually unknown outside of L. tredecimguttatus. The proposed study seeks to investigate the molecular and functional evolution of the latrotoxin gene family by characterizing its diversity across spider species. Comparative molecular analyses will also be coupled to functional studies of a-latrotoxin in order to identify the evolutionary steps leading to its vertebrate toxicity. The proposed study has three specific aims: (1) to characterize the diversity of latrotoxins in three focal species using genomic and proteomic approaches;(2) to compare the excitatory properties of a-latrotoxin from these three species by conducting functional assays of recombinant a-latrotoxins on vertebrate cells;and (3) to investigate the global phylogenetic distribution and molecular evolution of latrotoxins with bioinformatic analyses to pinpoint the timing and circumstances surrounding the origin of a-latrotoxin. Achieving these aims will provide fundamental information about this medically significant gene family, and will also establish the range of species that synthesize hazardous latrotoxins. Relevance: a-latrotoxin is an essential tool for the study of vertebrate neurotransmitter release. Characterization of related toxins will provide a wider breadth of molecular probes to more precisely determine the mechanistic basis of neuronal and glial signaling, aiding studies of neurological development and disorders. The proposed research will also allow biomedical researchers to synthesize antivenoms tailored to neutralize a-latrotoxin from different spider species;hospitalization due to a-latrotoxin envenomation is not uncommon and current therapies have reduced effectiveness at treating bites from certain species.